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Published on: July 17, 2013
Detection of methicillin-resistant Staphylococcus aureus persistence in osteoblasts using imaging flow cytometry
Dafne Bongiorno1, Nicolò Musso2, Lorenzo Mattia Lazzaro1
1Department of Biomedical and Biotechnological Sciences (BIOMETEC), Medical Molecular Microbiology and Antibiotic Resistance Laboratory (MMARLab), University of Catania, Catania, Italy.
Abstract:
Methicillin-resistant S. aureus has been reported as the main pathogen involved in chronic infections, osteomyelitis, and prosthetic joint infections. The host/pathogen interaction is dynamic and requires several changes to promote bacterial survival. Here, we focused on the internalization and persistence behavior of well-characterized Staphylococcus aureus invasive strains belonging to the main ST-MRSA-SCCmec clones. To overcome the limitations of the cell culture method, we comparatively analyzed the ability of internalization within human MG-63 osteoblasts with imaging flow cytometry (IFC). After evaluation by cell culture assay, the MRSA clones in the study were all able to readily internalize at 3h postinfection, the persistence of intracellular bacteria was evaluated at 24h both by routine cell culture and IFC assay, after vancomycin-BODIPY staining. A statistical difference of persistence was found in ST5-SCCmecII (26.59%), ST228-SCCmecI (20.25%), ST8-SCCmecIV (19.52%), ST239-SCCmecIII (47.82%), and ST22-SCCmecIVh (50.55%) showing the same ability to internalize as ATCC12598 (51%), the invasive isolate used as control strain for invasion and persistence assays. We demonstrated that the intracellular persistence process depends on the total number of infected cells. Comparing our data obtained by IFC with those of the cell culture assay, we obtained greater reproducibility rates and a number of intracellular bacteria, with the advantage of analyzing live host cells. Moreover, with some limitations related to the lack of whole-genome sequencing analysis, we validated the different proclivities to persist in the main Italian HA-MRSA invasive isolates and our results highlighted the heterogeneity of the different clones to persist during cell infection.
Insights
Methicillin-resistant Staphylococcus aureus (MRSA) clones show varied intracellular persistence in human osteoblasts. Imaging flow cytometry revealed significant differences in bacterial survival, highlighting clone-specific host-pathogen interactions crucial for chronic infections.
Area of Science:
- Microbiology
- Infectious Diseases
- Cell Biology
Background:
- Methicillin-resistant Staphylococcus aureus (MRSA) is a primary cause of persistent infections, including osteomyelitis and prosthetic joint infections.
- Understanding host-pathogen interactions is critical for developing effective treatments against MRSA.
- Specific MRSA strains, particularly those belonging to major ST-MRSA-SCCmec clones, exhibit distinct behaviors within host cells.
Purpose of the Study:
- To investigate and compare the internalization and intracellular persistence of well-characterized MRSA strains within human osteoblasts.
- To evaluate the efficacy of imaging flow cytometry (IFC) as an improved method for studying host-pathogen dynamics compared to traditional cell culture assays.
Main Methods:
- Human MG-63 osteoblasts were infected with various MRSA clones.
- Internalization was assessed at 3 hours post-infection.
- Intracellular bacterial persistence was evaluated at 24 hours post-infection using both routine cell culture and vancomycin-BODIPY stained IFC.
- Statistical analysis was performed to compare persistence rates among different MRSA clones.
Main Results:
- All tested MRSA clones demonstrated significant internalization into osteoblasts within 3 hours.
- Intracellular persistence varied significantly among MRSA clones at 24 hours, with ST239-SCCmecIII (47.82%) and ST22-SCCmecIVh (50.55%) showing higher rates.
- IFC provided greater reproducibility and allowed analysis of live host cells, offering advantages over standard cell culture methods.
- The study identified heterogeneity in the persistence capabilities of different HA-MRSA clones within host cells.
Conclusions:
- MRSA clones exhibit distinct intracellular persistence behaviors within human osteoblasts, influenced by the specific clone and the number of infected cells.
- Imaging flow cytometry is a robust and advantageous technique for quantifying intracellular bacterial persistence.
- These findings underscore the heterogeneity of MRSA clones and their potential to cause persistent infections, informing future therapeutic strategies.
